Abstract

The basolateral amygdala complex, which contains the lateral (LA) and basal (BA) subnuclei, is a critical substrate of associative learning related to reward and aversive stimuli. Auditory fear conditioning studies in rodents have shown that the excitation of LA pyramidal neurons, driven by the inhibition of local GABAergic interneurons, is critical to fear memory formation. Studies examining the role of the BA in auditory fear conditioning, however, have yielded divergent outcomes. Here, we used a neuron-specific chemogenetic approach to manipulate the excitability of mouse BA neurons during auditory fear conditioning. We found that chemogenetic inhibition of BA GABA neurons, but not BA pyramidal neurons, impaired fear learning. Further, either chemogenetic stimulation of BA GABA neurons or chemogenetic inhibition of BA pyramidal neurons was sufficient to generate the formation of an association between a behavior and a neutral auditory cue. This chemogenetic memory required presentation of a discrete cue, and was not attributable to an effect of BA pyramidal neuron inhibition on general freezing behavior, locomotor activity, or anxiety. Collectively, these data suggest that BA GABA neuron activation and the subsequent inhibition of BA pyramidal neurons play important role in fear learning. Moreover, the roles of inhibitory signaling differ between the LA and BA, with excitation of pyramidal neurons promoting memory formation in the former, and inhibition of pyramidal neurons playing this role in the latter.

Significance Statement

The basolateral amygdala complex, which consists of lateral (LA) and basal (BA) subnuclei, is a critical substrate of associative learning. Although inhibition of GABAergic interneurons and subsequent disinhibition of pyramidal neurons in the LA is critical to fear learning, the contribution of the BA is less clear. Here, we used a chemogenetic approach to manipulate pyramidal and GABA neuron excitability in the BA during fear conditioning. We found that BA GABA neuron activity is necessary for fear learning, and that BA GABA neuron stimulation or BA pyramidal neuron inhibition can induce an association between a behavior and an auditory cue. These findings expand our understanding of the fear learning circuitry and highlight a novel role for inhibitory signaling.

Introduction

Abnormal associative learning is a hallmark of many mental health disorders, including obsessive compulsive disorder, post-traumatic stress disorder, and drug addiction (Nielen et al., 2009; Maren et al., 2013; Bowers and Ressler, 2015; Taylor and Torregrossa, 2015). These disorders are characterized by the aberrant establishment of associations between two stimuli or a stimulus and behavior, or the failure to extinguish associations that are no longer relevant. This can result in exaggerated or inappropriate emotional responses, as well as prolonged maladaptive behaviors that persist despite negative consequences. Understanding the neuronal mechanisms that support the formation and durability of associative memories is key to developing effective therapies for diseases in which associative learning is disrupted.

Materials and Methods

Animals

Animal experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee. CaMKIICre [B6.Cg-Tg(Camk2a-cre)T29-1Stl/J] and GADCre (B6N.Cg-Gad2tm2(cre)Zjh/J) lines were purchased from The Jackson Laboratory, and have been maintained by backcrossing against the C57BL/6J strain. Offspring from these crosses were used to generate the Cre(+) and Cre(−) mice used in this study. The generation of conditional CaMKIICre(+):Girk1fl/fl mice was described previously (Marron Fernandez de Velasco et al., 2017). Unless specifically noted, males and females were used in all experiments, and groups were balanced by sex. All mice were maintained on a 12 h light/dark cycle, and were provided ad libitum access to food and water.

Reagents

Intracranial viral manipulations

AAV8-hSyn-DIO-hM4Di-mCherry and AAV8-hSyn-DIO-hM3Dq-mCherry were purchased from the UNC Vector Core. Mice (7-8 weeks old) were placed in a stereotaxic device (David Kopf Instruments) under isoflurane anesthesia. Microinjectors were made by affixing a 33-gauge stainless steel hypodermic tube within a shorter 26-gauge stainless steel hypodermic tube. The microinjectors were attached to polyethylene-20 tubing affixed to 10 μl Hamilton syringes, and were lowered through burr holes in the skull to the BA (from bregma: −1.65 mm A/P, ±3.25 mm M/L, −4.7 mm D/V) or LA (from bregma: −1.6 mm A/P, ±3.3 mm M/L, −4.2 mm D/V); 500 nl (4–7 × 1012 viral particles/ml) of viral solution per side was injected over 5 min. The syringe was left in place for 10 min following infusion to reduce solution backflow along the infusion track. Subsequent electrophysiological and behavioral experiments were performed 4 weeks after surgery to allow for full recovery and viral expression. The scope and accuracy of viral targeting was assessed by tracking viral-mediated mCherry fluorescence in serial coronal sections Cre(+) mice. Fluorescence was observed along the full rostrocaudal axis of the BLA complex. Only data from mice in which the majority (>80%) of expression was confined to the targeted subregion (LA or BA), with limited or no diffusion to adjacent structures (i.e., central amygdala or cortex), were analyzed.

Slice electrophysiology

Coronal slices (270–280 μm) containing the BLA complex were prepared from mice (5–12 weeks), as described previously (Arora et al., 2010; Hearing et al., 2013; Marron Fernandez de Velasco et al., 2017), and were incubated at 32°C in ACSF for >30 min before recording. All measured and command potentials factored in a junction potential (−15 mV) predicted using JPCalc software (Molecular Devices). Agonist-induced somatodendritic currents were measured in an ACSF bath using a K-gluconate pipette solution, at a holding potential (Vhold) of −60 mV. Holding current, input resistance, and series resistance values were monitored during each experiment by tracking responses to periodic (0.2 Hz) voltage steps (−5 mV, 800 ms). Only experiments with stable (<20% variation) and low series resistances (<30 MΩ) were analyzed. For rheobase assessments, cells were held in current-clamp mode and given 1 s current pulses, beginning at −60 pA and progressing in 20 pA increments until spiking was elicited. Miniature IPSCs (mIPSCs) were recorded (Vhold= −70 mV) for 1.5 min using a 140 mm CsCl-based pipette solution, with 2 mm kynurenic acid and 0.5 μm TTX present in the bath to block ionotropic glutamatergic activity and action potentials, respectively. mIPSCs were analyzed with Minianalysis software (Synaptosoft), using a 10 pA detection threshold. All electrophysiological datasets include data from at least two offspring from different breeder pairs.

Behavioral testing

Adult mice (8–12 weeks) were evaluated using established delay fear conditioning (Tipps et al., 2014) and elevated plus maze (EPM) tasks (Victoria et al.,
2016). For fear conditioning experiments, the CS was a 65 dB white noise (30 s) and the US was a 0.5 mA footshock (2 s), administered during the last 2 s of the CS presentation. Before training, mice were exposed to the fear conditioning room and pre-handled for 2 d to acclimate animals to the behavioral room and investigator. Four conditioning protocols were used in this study: (1) 3 CS/3 US: mice were exposed to three CS–US pairings, with CS presentations separated by 90 s (7.5 min total); (2) 3 CS/0 US: mice were exposed to the CS three times using the same timing sequence as with the 3 CS/3 US protocol, but no US was delivered (7.5 min total); (3) 0 CS/0 US: mice were placed in the chambers but were not exposed to either CS or US (7.5 min total); and (4) 1 CS/1 US: mice were exposed to a single CS–US pairing, preceded and followed by 90 s intervals (3.5 min total). To assess context learning (24 h after training), mice were returned to the conditioning chambers and freezing was evaluated for 5 min. To assess cue learning (48 h after training), chambers were reconfigured using a white plastic insert to cover the bar floor and a black tent insert to alter the size, shape, and color of the chambers. Inserts were cleaned with 0.1% acetic acid to provide a distinct olfactory cue. Freezing was monitored throughout the 15 min cue recall test period, divided into 5 × 3 min bins that included 2 × 3 min CS presentations (Tipps et al., 2014). Freezing behavior, along with measures of average and maximum motion, was assessed automatically using Video Freeze v2.6.1.72 software (Med Associates). For EPM studies, time spent in open and closed arms, and number of arm entries, were recorded for 5 min, beginning when the mouse made its first entry into any arm. EPM performance was scored manually, by an experienced investigator.

Data analysis

Data are presented throughout as the mean ± SEM. Statistical analyses were performed using Prism 6 (GraphPad Software) and SigmaPlot 11.0 (Systat Software). Sex was included as a variable in initial analyses. Because no impact of sex was observed in any study, all data from male and female subjects were pooled. Pooled data were analyzed by Student’s t test or repeated-measures ANOVA, as appropriate. Pairwise comparisons were performed using Bonferroni or Holms–Sidak (H–S) tests, when appropriate. For all statistical comparisons, differences were considered significant if p < 0.05.

To test the efficacy of the neuron-specific chemogenetic approach, we first examined whether chemogenetic inhibition of LA pyramidal neurons could disrupt auditory fear learning. Previous work has shown that optogenetic inhibition of LA pyramidal neurons disrupted auditory fear conditioning (Johansen et al., 2014), a key line of evidence supporting the contention that LA pyramidal neuron excitation is critical to fear learning. We targeted the LA of CaMKIICre(+) mice with an AAV8-hSyn-DIO-hM4Di-mCherry virus, and observed robust hM4Di expression 4 weeks later (Fig. 2A,B). Application of the hM4Di agonist CNO (10 μm) to acutely isolated slices from these mice reduced the excitability (i.e., increased the rheobase) of hM4Di-expressing LA pyramidal neurons (Fig. 2C,D). We next evaluated viral-treated CaMKIICre(+) and CaMKIICre(-) littermates in an amygdala-dependent delay fear conditioning protocol involving three pairings of an auditory cue/CS and footshock/US (3 CS/3 US; Fanselow and LeDoux, 1999; LeDoux, 2000; Nonaka et al., 2014). All subjects received CNO (2 mg/kg, .i.p.) 30 min before training, to promote inhibition of hM4Di-expressing neurons during the early (acquisition and consolidation) stages of fear learning (Roth, 2016). Context and cue recall testing occurred 24 and 48 h after training, respectively, in the absence of CNO. All subjects received the same viral and CNO treatments, and because CNO was only administered before training, any phenotypes observed during recall tests were interpreted as reflecting an impact of the neuron-specific manipulation on long-term fear memory formation. As predicted based on previous optogenetic inhibition experiments (Johansen et al., 2014), chemogenetic inhibition of LA pyramidal neurons during training impaired fear memory formation, as evidenced by decreased freezing during both the context and cue recall tests (Fig. 2E). Using the same approach to target BA pyramidal neurons in CaMKIICre(+) mice (Fig. 2F,G), we observed that CNO also reduced the excitability (increased the rheobase) of hM4Di-expressing BA pyramidal neurons (Fig. 2H,I). Chemogenetic inhibition of BA pyramidal neurons. Although the CaMKIICre(+) mice showed increased freezing to the third CS presentation during training, there was no difference in recall for either the auditory cue or associated context in the 3 CS/3 US paradigm (Fig. 2J). Thus, inhibition of BA pyramidal neurons during training did not impair long-term memory formation.

Chemogenetic inhibition of pyramidal neurons in the LA, but not BA impairs auditory fear conditioning. A, hM4Di-mCherry fluorescence in the LA of a CaMKIICre(+) mouse, 4 weeks after infusion of AAV8-hSyn-DIO-hM4Di-mCherry. Scale bar, 50 μm. B, Schematic summarizing the distribution of hM4Di-mCherry fluorescence in the LA (spanning –1.22mm to –1.94mm posterior from bregma) of CaMKIICre(+) mice evaluated in E. C, CNO-induced inhibition of an hM4Di-expressing LA pyramidal neuron. Rheobase was measured before (baseline) and after application of CNO (10 μm). The current-step protocol is depicted below the traces. The first step to elicit spiking is highlighted in red; the trace shown is the response to the denoted current step. D, Rheobase summary for hM4Di-expressing LA pyramidal neurons, before and after CNO application (t(3)=4.4, *p = 0.021). Each experiment is shown as connected circles (n = 4). E, Impact of LA pyramidal neuron inhibition on auditory fear conditioning. CaMKIICre(+)/hM4Di (black) and CaMKIICre(−)/hM4Di (white) mice were trained using a 3 CS/3 US paradigm. CNO (2 mg/kg, i.p.) was administered 30 min before training. Freezing behavior during training is plotted on the left. The yellow bars represent CS presentations, and the red bars represent US presentations. There was no main effect of genotype (F(1,54)=2.6, p = 0.14). The plots on the right show freezing during context (t(9)=4.0, **p = 0.003) and cue (t(9)=2.9, *p = 0.017) recall tests. The bars represent the mean ± SEM, with dots next to the bars denoting individual data points (n = 5–6/group). Sex differences were not assessed for this experiment. F, hM4Di-mCherry fluorescence in the BA of a CaMKIICre(+) mouse, 4 weeks after infusion of AAV8-hSyn-DIO-hM4Di-mCherry. Scale bar, 50 μm. G, Schematic summarizing the distribution of hM4Di-mCherry fluorescence in the BA (coronal view, spanning –1.22mm to –1.94mm posterior from bregma) of CaMKIICre(+)/hM4Di mice evaluated in C. H, CNO-induced inhibition of an hM4Di-expressing BA pyramidal neuron. Rheobase was measured before (baseline) and after application of CNO (10 μm), as described for C. I, Rheobase summary for hM4Di-expressing BA pyramidal neurons, before and after CNO application (t(6)=7.6, ***p < 0.001). Each experiment is shown as connected circles (n = 7). J, Impact of BA pyramidal neuron inhibition on fear learning. CaMKIICre(+)/hM4Di (red) and CaMKIICre(−)/hM4Di (white) mice were trained using a 3 CS/3 US paradigm. CNO (2 mg/kg, i.p.) was administered 30 min before training. Freezing behavior during training is plotted on the left. The yellow bars represent CS presentations, and the red bars represent US presentations. There was a significant main effect of genotype (F(1,132)=4.9, p = 0.038; CS3: ***p < 0.001, post-CS3: *p = 0.023). The plots on the right show freezing during context (t(22)=1.9, p = 0.074) and cue (t(29)=0.1, p = 0.905) tests. Error bars represent the mean ± SEM, with dots next to the bars denoting individual data points (n = 5/6–7/5 males/females per group).

The ability of LA pyramidal neuron inhibition to impair memory formation is consistent with previous reports; however, the lack of effect of BA pyramidal neuron inhibition on fear learning suggests that inhibitory signaling may play a different role in this region. To probe the impact of local GABA-mediated inhibitory signaling within the BA on fear learning, we expressed hM4Di in the BA of GADCre(+) mice (Fig. 3A). As expected, CNO reduced the excitability (increased the rheobase) of hM4Di-expressing BA GABA neurons in slices from GADCre(+) subjects (Fig. 3B,C). In contrast to our results in BA pyramidal neurons, chemogenetic inhibition of BA GABA neurons during training significantly impaired fear memory formation, as illustrated by reduced freezing during the context and cue recall tests (Fig. 3D). Thus, GABA neuron activity in the BA is required for the acquisition of fear memory.

Chemogenetic inhibition of BA GABA neurons impairs auditory fear conditioning. A, Schematic summarizing the distribution of hM4Di-mCherry fluorescence in the BA (coronal view, spanning –1.22mm to –1.94mm posterior from bregma) of GADCre(+)/hM4Di mice evaluated in D. B, CNO-induced inhibition of an hM4Di-expressing BA GABA neuron. Rheobase was measured before (baseline) and after application of CNO (10 μm). The current-step protocol is depicted below the traces. The first step to elicit spiking is highlighted in blue; the trace shown is the response to the denoted current step. C, Rheobase summary for hM4Di-expressing BA GABA neurons, before and after CNO application (t(4)=3.2, *p = 0.033). Each experiment is shown as connected circles (n = 5). D, Impact of BA GABA neuron inhibition on fear learning. GADCre(+)/hM4Di (blue) and GADCre(−)/hM4Di (white) mice were trained with a 3 CS/3 US paradigm. CNO (2 mg/kg, i.p.) was given 30 min before training. Freezing during training is shown on the left. The yellow bars denote CS presentations and the red bars indicate US presentations. There was no main effect of genotype (F(1,174)=0.1, p = 0.71). The plots on the right show freezing during context (t(29)=4.9, ***p < 0.001) and cue (t(29)=4.5,***p < 0.001) tests. Error bars represent the mean ± SEM, with dots next to the bars denoting individual data points (n = 6/8–9/8 males/females group).

Chemogenetic stimulation of BA GABA neurons promotes fear learning

Previous work has shown that direct optogenetic stimulation of LA pyramidal neurons during presentation of a neutral auditory cue, in the absence of a footshock, is sufficient to generate a fear-like response to subsequent presentation of the cue (Johansen et al., 2010). Given the similar disruptive impact of chemogenetic inhibition of LA pyramidal (Fig. 2E) and BA GABA neurons (Fig. 3D) on fear learning, we next asked whether BA GABA neuron stimulation could also generate a learned response to an auditory cue. To test this, we injected a Cre-dependent excitatory chemogenetic virus (AAV8-hSyn-DIO-hM3Dq-mCherry) into the BA of GADCre(+) mice (Fig. 4A). As expected, the application of CNO increased the excitability (reduced the rheobase) of hM3Dq-expressing BA GABA neurons (Fig. 4B,C).

BA GABA neuron stimulation generates an association between a behavior and an auditory cue. A, Schematic summarizing the distribution of hM3Dq-mCherry fluorescence in the BA (coronal view, spanning –1.22mm to –1.94mm posterior from bregma) of the GADCre(+)/hM3Dq mice evaluated in D–F. B, CNO-induced stimulation of an hM3Dq-expressing BA GABA neuron. Rheobase was measured before (baseline) and after application of CNO (10 μm). The current-step protocol is depicted below the traces. The first step to elicit spiking is highlighted in blue; the trace shown is the response to the denoted current step. C, Rheobase summary for hM3Dq-expressing BA GABA neurons, before and after CNO application (t(6)=4.4, **p < 0.001). Each experiment is represented as connected circles (n = 7). D, Impact of BA GABA neuron stimulation during training. Freezing behavior during training (3 CS/0 US) for GADCre(+)/hM3Dq (blue) and GADCre(−)/hM3Dq (white) mice. CNO (2 mg/kg, i.p.) was given 30 min before training. Yellow bars represent CS presentations. There was a significant main effect of genotype (F(1,90)=6.5, p = 0.020; CS3: **p = 0.005, post-CS3: ***p < 0.001). E, Freezing during the context recall test. The plot on the left shows freezing for each 1 min bin of the 5 min context test. There was a significant main effect of genotype (F(1,60)=7.9, p = 0.013; bin 1: *p = 0.01, bin 5: *p = 0.04). The plot on the right shows the mean percentage freezing for the entire context test (t(15)= −2.8, *p = 0.013). F, Freezing during the cue recall test. The plot on the right shows freezing during each 3 min bin of the cue test, with CS presentations highlighted in yellow. There was a significant main effect of genotype (F(1,75)=13.6, p < 0.001; CS1: **p = 0.008, CS2: *p = 0.03). GADCre(+)/hM3Dq (t(14)= −2.6, p = 0.021), but not GADCre(−)/hM3Dq mice (t(16)=−1.5, p = 0.152) froze significantly more during the CS periods than the non-CS periods. The bars on the right represent mean percentage freezing during the combined CS presentations (t(15)=−2.5, *p = 0.023). Error bars in C, E, and F represent the mean ± SEM, with dots next to the bars denoting individual data points (n = 4/4–5/4 males/females per group).

To assess the behavioral impact of this manipulation, we used a modified fear conditioning protocol in which subjects were given three presentations of an auditory cue/CS, without footshock/US (3 CS/0 US; Tipps et al., 2014). BA GABA neuron stimulation during training yielded increased freezing to the third CS presentation (CS3), relative to controls (Fig. 4D). Significantly enhanced freezing was also seen in GADCre(+)/hM3Dq subjects during the subsequent context and cue recall tests, conducted in the absence of CNO (Fig. 4E,F). Importantly, no group differences in freezing behavior were observed before the first CS presentation during training (Fig. 4C), or in response to the altered context during the cue recall test (Fig. 4F). Further, GADCre(+)/hM3Dq mice froze significantly more during the CS presentations during the cue test compared to the non-CS periods, whereas GADCre(−)/hM3Dq subjects did not. These findings suggest that BA GABA neuron stimulation does not elicit a general increase in freezing behavior, but rather, promotes the formation of an associative response to an otherwise neutral auditory cue and associated context.

Given that BA GABA neuron stimulation is sufficient to generate a long-term associative response to a neutral auditory cue, and that BA GABA neuron stimulation inhibits BA pyramidal neurons, we next asked whether the hM4Di-mediated inhibition of BA pyramidal neurons during training could also induce the formation of a long-term behavioral response to a neutral auditory cue. We again used CaMKIICre(+) mice and an AAV8-hSyn-DIO-hM4Di-mCherry virus to express hM4Di in BA pyramidal neurons (Fig. 6A), and administered CNO to viral-treated CaMKIICre(+) and CaMKIICre(−) subjects 30 min before training in the 3 CS/0 US paradigm. Similar to the outcome of the BA GABA neuron stimulation experiments, BA pyramidal neuron inhibition promoted freezing to the second and third CS presentations during training, but had no effect on freezing before the first CS presentation (Fig. 6B). Moreover, BA pyramidal neuron inhibition during training resulted in the formation of a long-term associative response, as revealed by increased freezing relative to control subjects in both the context and cue recall tests (Fig. 6C,D). Importantly, no group differences were observed on initial introduction of the subjects to the altered context during the cue test (Fig. 6D). Further, CaMKIICre(+)/hM4Di mice, but not CaMKIICre(−)/hM4Di mice, froze significantly more during the CS presentations during the cue test compared to the non-CS periods. These findings suggest that chemogenetic inhibition of BA pyramidal neurons does not elicit a general increase in freezing behavior, but rather, supports the formation of an associative response to an otherwise neutral auditory cue and associated context.

Although these data suggest that BA pyramidal neuron inhibition is a key contributor to associative memory formation, we designed a series of control experiments to test alternative explanations. First, although our study design ensured that all animals received the same viral construct and CNO treatment, a baseline difference in fear learning between CaMKIICre(−) and CaMKIICre(+) littermates could explain our observations. There was no difference in freezing behavior, however, during training or recall tests for untreated CaMKIICre(+) and CaMKIICre(−) littermates trained with the 3 CS/0 US protocol, in the absence of CNO (Fig. 7A). Second, although freezing levels were similar across groups before the initial CS presentation during training, and on initial exposure to the altered context during the cue test, we conducted additional tests to determine if chemogenetic inhibition of BA pyramidal neurons resulted in locomotor impairments and/or nonspecific freezing behavior. Chemogenetic inhibition of BA pyramidal neurons had no impact on freezing behavior or the average motion index or maximum motion measures during training using a 0 CS/0 US paradigm (context exposure only; Fig. 7B). Freezing behavior in the subsequent recall tests was also unaffected (Fig. 7B). In addition to demonstrating that chemogenetic inhibition of BA pyramidal neurons does not evoke a nonspecific increase in freezing behavior (or a nonspecific decrease in locomotion), these data also show that the learned association generated by BA pyramidal neuron inhibition requires a discrete CS presentation.

Induction of memory formation by BA pyramidal neuron inhibition is not because of genotype differences, nonspecific freezing behavior, or anxiety changes. A, Genotype difference test. Training with the 3 CS/0 US protocol in untreated CaMKIICre(+) (black) and CaMKIICre(−) (white) mice is shown on the left. Yellow bars represent the CS presentations. There was no effect of genotype (F(1,66)=0.37, p = 0.55). Freezing during context (t(11)=−0.4, p = 0.73) and cue (t(11)=0.4, p = 0.69) recall tests for untreated CaMKIICre(+) and CaMKIICre(−) littermates, following conditioning with the 3 CS/0 US protocol, is plotted on the right. Sex effects were not analyzed for this study (n = 5–8/genotype). B, Nonspecific freezing behavior test. Training with the 0 CS/0 US protocol in CaMKIICre(+)/hM4Di (red) and CaMKIICre(−)/hM4Di (white) mice is shown on the left. CNO (2 mg/kg, i.p.) was given 30 min before training. No CS or US presentations were given. The plot on the left show freezing during training. No genotype differences were seen during training (F(1,168)=0.5, p = 0.49). The plots on the right show freezing during context (t(28)=−0.0, p = 0.98) and cue (t(28)=−0.5, p = 0.65) recall tests (n = 6/6–8/10 males/females per group). No genotype differences were detected in average motion (F(1,140)=0.9, p = 0.35) or maximum motion (F(1,140)=0.95, p = 0.34) indices (data not shown). C, One CS/US protocol test. Freezing following training with a weak (1 CS/1 US) fear conditioning protocol in CaMKIICre(+)/hM4Di (red) and CaMKIICre(−)/hM4Di (white) mice is shown on the left. CNO (2 mg/kg, i.p.) was given 30 min before training. The yellow bar represents the CS presentation and the red bar represents the US presentation. There was no effect of genotype (F(1,50)=0.83, p = 0.37). The plots on the right show freezing during context (t(25)=1.0, p = 0.35) and cue (t(25)=−0.8, p = 0.46) recall tests (n = 7/6–7/7 males/females per group). D, Anxiety test. Percentage of time spent in the open arms (D; t(23)=0.1, p = 0.94) by CaMKIICre(+)/hM4Di (red) and CaMKIICre(−)/hM4Di (white) mice and total number of arm entries (E; t(23)=1.1, p = 0.28). All subjects received CNO (2 mg/kg, i.p.) 30 min before EPM testing (n = 5/5–8/7 males/females group). Bars represent the mean ± SEM, with dots next to each bar denoting the individual data points.

The dependence of the CNO/hM4Di-induced fear memory on CS presentation raised the possibility that BA pyramidal neuron inhibition during training may simply strengthen an otherwise weak association between the US and auditory CS. To test this prospect, we used a weak conditioning protocol involving a single CS–US pairing (1 CS/1 US), which would allow us to detect an increase in freezing behavior that might be obscured in more robust standard (3 CS/3 US) conditioning protocol. BA pyramidal neuron inhibition during conditioning in the 1 CS/1 US protocol did not alter freezing during training or enhance context or cue-induced freezing in recall tests (Fig. 7C). Thus, although BA pyramidal neuron inhibition may induce memory formation, it does not determine the strength of the association when a US is presented.

Finally, as anxiety can impact performance in fear conditioning tests (Izquierdo et al., 2016), we asked whether BA pyramidal neuron inhibition altered anxiety-related behavior, using the EPM test. BA pyramidal neuron inhibition did not alter time spent in the open arms of the EPM or number of total (open + closed) arm entries (Fig. 7D), indicating that BA pyramidal neuron inhibition does not impact anxiety-related behavior or general motor activity. Collectively, these data also suggest that inhibition of BA pyramidal neurons does not promote a subjective sense of fear, which would be expected to generate increased freezing in both the 0 CS/0 US and 1 CS/1 US conditions, and potentially decrease the time spent in the open arms of the EPM.

To test whether GIRK channel activation is also required for the chemogenetic induction of associative learning, we evaluated CaMKIICre(+):Girk1fl/fl mice in the 3 CS/0 US conditioning protocol, 4 weeks after intra-BA infusion of AAV8-hSyn-DIO-hM4Di-mCherry virus. Mice were randomly assigned to receive saline or CNO 30 min before training. The levels of freezing observed during training, and in subsequent context and cue recall tests, did not differ between saline- and CNO-treated subjects (Fig. 8D). Thus, the loss of GIRK channels, a primary mediator of hM4Di influence in BA pyramidal neurons, is sufficient to block the generation of an associative behavioral response via chemogenetic inhibition of BA pyramidal neurons.

Discussion

Inhibitory interneurons regulate the activity of local excitatory principal neurons in many brain regions. These interneurons are critical for shaping network activity and exhibit experience-induced plasticity, suggesting that changes in inhibitory signaling may underlie long-term learning and behavioral changes (Lucas and Clem, 2018). Within the field of learning and memory, the impact of inhibitory interneuron activity is well illustrated by the disinhibition model of associative fear memory formation (Letzkus et al., 2015). In this model, the combined effect of exposure to a CS and US during training results in an overall decrease in GABAergic input to glutamatergic projection neurons, leading to an increase in projection neuron excitability. Examples of disinhibition-based signaling can be found in several brain regions, including the cortex (Letzkus et al., 2011) and BLA complex (Wolff et al., 2014).

Available data support the contention that excitation of LA pyramidal neurons is a critical step in auditory fear conditioning. For example, optogenetic stimulation of LA pyramidal neurons in mice can generate or enhance fear memory formation (Johansen et al., 2010; Yiu et al., 2014), whereas optogenetic (Johansen et al., 2014) or chemogenetic (Fig. 2) inhibition of LA pyramidal neurons impairs fear learning. In addition, the excitability of LA pyramidal neurons correlates positively with inclusion of those neurons in the subsequent fear memory trace (Zhou et al., 2009; Kim et al., 2014). Consistent with a key role for pyramidal neuron excitation in fear memory formation, the combined effect of exposure to a CS and US during fear conditioning results in an overall decrease in GABAergic input to pyramidal neurons of the BLA complex, resulting in pyramidal neuron disinhibition (Wolff et al.,
2014). Indeed, manipulations of the parvalbumin-expressing subtype of GABA neurons in the BLA complex have been shown to modulate the strength of associative memory formation in response to a footshock, with inhibition of parvalbumin-expressing neurons during the CS–US presentation increasing fear learning and stimulation of parvalbumin-expressing neurons impairing fear learning (Wolff et al., 2014).

While studies have consistently illustrated the role of LA pyramidal neuron excitation in auditory fear conditioning, most of the published lesion and pharmacological inhibition studies have not supported a role for the BA in the acquisition of cue fear memories (Nader et al., 2001; Anglada-Figueroa and Quirk, 2005; Calandreau et al., 2005; Onishi and Xavier, 2010; Akagi Jordão et al., 2015). However, given that lesions and pharmacologic inhibitors lack neuronal specificity and can have broad and nonspecific effects on neural circuits, we revisited the role of the BA in auditory fear learning using a neuron-specific chemogenetic approach. We found that activation of BA GABA neurons and inhibition of BA pyramidal neurons are critical steps in the formation of associative fear memories. Given that optogenetic stimulation of LA principal neurons (Johansen et al., 2014), and chemogenetic inhibition of BA pyramidal neurons (Fig. 6), can both promote the formation of learned response to a neutral auditory cue, our data suggest the intriguing prospect that the fear conditioning-induced disinhibition of LA pyramidal neurons triggers the stimulation of BA GABA neurons and feedforward inhibition of BA pyramidal neurons.

This conceptual framework can reconcile apparently contradictory prior observations related to the impact of lesions, as well as pharmacologic and genetic interventions, on auditory fear conditioning. For example, the lack of impact of pharmacological inactivation of the BA during auditory fear conditioning was interpreted as evidence that the BA is not required for fear learning (Calandreau et al., 2005). Given that chemogenetic inhibition of BA pyramidal neurons also did not preclude fear learning (Fig. 2), we speculate that the potentially detrimental effect on fear learning of inhibiting BA GABA neurons using a broad pharmacological approach might be offset by the direct inhibition of BA pyramidal neurons. Similarly, a system in which LA pyramidal neuron inhibition significantly impairs long-term memory formation, whereas BA pyramidal neuron inhibition does not, could explain why some manipulations of pyramidal neurons across the entire BLA complex during training resulted in significant impairments observed during recall testing (Wolff et al., 2014), whereas others did not (Namburi et al., 2015). Our results show that inhibitory manipulations of pyramidal neurons within the BLA complex that bias toward the LA would be expected to impair fear memory formation, whereas the same manipulation primarily targeted within the BA would not. A more extensive analysis of these manipulations in the LA will be needed to confirm this hypothesis, however. The work presented here focuses on the BA, primarily based on our initial finding that pyramidal inhibition in the LA produced the anticipated impairment in fear memory formation, whereas BA pyramidal neuron inhibition did not. Although our investigation of this interesting distinction yielded novel results, the application of our approach to the LA may also yield surprising effects, and will be an important direction for future work.

Our data suggest an interesting extension of the circuitry and signaling mechanisms implicated in auditory fear learning; however, limitations associated with the approaches used in this study are worth noting. First, although the CaMKIICre and GADCre transgenic mouse lines used in this study facilitated the manipulation of distinct neuron populations, pyramidal and GABA neurons in the BLA complex are diverse. The GADCre line, for example, drives expression in all major GABA neuron subtypes (Taniguchi et al., 2011), including the PV and somatostatin subtypes that exert opposing influence on LA pyramidal neuron activity (Lovett-Barron et al., 2014; Wolff et al., 2014; Lucas and Clem, 2018). Thus, extending our efforts to identify the relevant subpopulation(s) of inhibitory interneurons that are essential for auditory fear conditioning will be informative. Similarly, the afferent and efferent connections of pyramidal neurons in the BLA complex are diverse, and these distinctions have significant implications for both conditioned fear and anxiety (Janak and Tye, 2015; Beyeler et al., 2016, 2018; Burgos-Robles et al., 2017). Thus, it will be interesting to use projection-specific chemogenetic manipulations of discrete BA microcircuits to understand which projection(s) is/are most relevant to the facilitation of associative learning by inhibitory signaling reported in this study.

We also note that the time course of the chemogenetic manipulations used in this study encompassed most of the acquisition and consolidation periods (Roth, 2016). Although this design allowed us to probe the role of inhibitory signaling without an a priori assumption regarding when such signaling might be relevant, future work using a more temporally discrete approach will be needed to identify critical time points within this period for the inhibitory mechanisms identified in this study. A more temporally restricted approach will also allow for additional control measures, such as the inclusion of a CS presented in the absence of neuronal inhibition, to further validate the specificity of our reported effects. On a related front, although our work focuses on the acquisition of new fear memories, the BA has been implicated in the recall and extinction of established fear memories (Herry et al., 2008; Amano et al., 2011), and the implications of our findings to the role played by this brain region in other aspects of fear learning is unclear.

It is also important to note that pyramidal neurons in the BLA complex also receive excitatory input during CS and US presentations (Letzkus et al., 2015). Our demonstration that BA pyramidal neuron inhibition promotes the association between an auditory cue and a behavioral response does not rule out a role for BA pyramidal neuron excitation in associative learning as well. Indeed, the inability of BA pyramidal neuron inhibition to generate a long-term memory in the absence of a discrete CS, and the lack of impact of BA pyramidal neuron inhibition in a weak fear conditioning paradigm (Fig. 7), suggest that other signals shape the resulting fear memory.

Together, our findings demonstrate that inhibitory signaling in the amygdala plays a more diverse and nuanced role in associative learning than originally thought. In combination with previous studies, our work shows that the cellular mechanisms underlying fear learning differ in the LA and BA, with pyramidal neuron excitation promoting memory formation in the former, and pyramidal neuron inhibition serving this role in the latter. It will be important to investigate more extensively the potential role of inhibitory signaling in normal associative learning processes, and in diseases in which these processes are disrupted.

Footnotes

The authors declare no competing financial interests.

This work was supported by NIH Grants to M.T. (AA025978) and K.W. (MH061933, DA034696). We thank Nicholas Carlblom for his exceptional care of the mouse colony and Zhillian Xia for all genotyping work.

KrettekJE, PriceJL (1978) A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid axonal connections. J Comp Neur178:255–280.doi:10.1002/cne.901780205pmid:627626

Synthesis

Reviewing Editor: Karen Szumlinski, University of California at Santa Barbara

Decisions are customarily a result of the Reviewing Editor and the peer reviewers coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewer(s) agreed to reveal their identity: Francisco Sotres-Bayon. Note: If this manuscript was transferred from JNeurosci and a decision was made to accept the manuscript without peer review, a brief statement to this effect will instead be what is listed below.

After consultation with the two original reviewers of your transferred article, we have reached the decision of “Revise-Editorial Review Only”. This decision was based on the acknowledgement that the authors have toned down their conclusions and provided appropriate discussion of the pitfalls and short-comings of their study. While both reviewers would like to see the present set of results followed up in future experiments, they are understanding of the labortory's situation and are willing to make concessions about the inclusion of more data. This being said, both reviewers have remaining concerns that linger from the previous round of comments that I will review for inclusion what we hope to be the final revision to this report. Please find the specific comments of the reviewers below:

Reviewer 1:

The authors have made some changes but are not fully responsive to prior critiques.

1. The authors continue to insist that there are no acquisition effects in Figure 2J by arguing that the inhibition of BA pyramidal neurons had no effect on the acquisition of *long-term* memories (lines 261-262). I do not understand this response. In truth, there is no effect on the retention but there is clearly an effect on acquisition. ‘Acquisition’, as used in standard learning and memory work, refers to the initial learning period, which is the left-side panel of Figure 2J. The authors show asterisks for CS3 and the period immediately after, meaning there is an effect on acquisition -- even if this difference did not alter the retention. I would suggest the authors correct this issue in the manuscript.

2. I appreciate the authors' inclusion of additional points in the Discussion, particularly regarding gating. However, the way authors use ‘gating’ and the way Luthi and others use the term are different. For Luthi (e.g., the Wolff 2014 paper the authors repeatedly cite), gating plays a permissive or enabling role for the synaptic plasticity underlying the memory formation. Thus, gating mechanisms can be manipulated to either enhance or impair the memory formation. Wolff et al. (2014) use CS-US pairings throughout the experiments (in contrast to the effects the authors of this manuscript find without a US) and demonstrate that PV neuron manipulations can enhance and impair the memory. Luthi's group argues that the PV neurons play a role in gating the CS-US associations.

In contrast, the authors of the present manuscript demonstrate that activation of BA neurons in the absence of a US can create a memory, yet such activation has no effect on a single CS-US pairing. At minimum, if this ‘gating’ were like Luthi's gating, such activation would enhance the memory formation of the single CS-US pairing. Considering the novelty of the authors' findings, the authors do themselves (and their findings) a disservice by suggesting that their findings are of the same kind as Wolff et al. The authors' findings are different and consequently rather interesting. I would suggest the authors refrain from referring to their findings as ‘gating’ or, at minimum, suggest that, if this is gating, it is different from what Wolff found in their work.

Reviewer 2:

This timely study with relevant findings extend an important literature on the study of associative learning mechanisms by highlighting a novel role for inhibitory signaling in the amygdala.

In this revised version, the authors addressed the concerns raised by making relevant changes to the text of the manuscript and figures. This changes included toning down the interpretations of their findings and thereby changing the title. Since there is no direct evidence for gating mechanism I suggest the authors mention this concept to a minimum.

Author Response

We would like to thank the reviewers and the editor for their helpful comments. We deeply appreciate the time the editor in particular has dedicated to the review process. Our response to the minor comments from the reviewers are given below in red. Likewise, the corresponding changes to the manuscript are highlighted in red in the main article file.

Reviewer 1:

The authors have made some changes but are not fully responsive to prior critiques.

1. The authors continue to insist that there are no acquisition effects in Figure 2J by arguing that the inhibition of BA pyramidal neurons had no effect on the acquisition of *long-term* memories (lines 261-262). I do not understand this response. In truth, there is no effect on the retention but there is clearly an effect on acquisition. ‘Acquisition’, as used in standard learning and memory work, refers to the initial learning period, which is the left-side panel of Figure 2J. The authors show asterisks for CS3 and the period immediately after, meaning there is an effect on acquisition -- even if this difference did not alter the retention. I would suggest the authors correct this issue in the manuscript.

While the acquisition period is a critical portion of the learning process, differences in this period do not necessarily reflect changes in learning. Auditory startle, footshock sensitivity, and many other factors can lead to significantly different freezing levels during acquisition that do not translate into changes in the resulting memory. As such, the learning field as a whole does not rely on differences during acquisition to define changes in learning/memory. Indeed, most papers do not even provide training data, because differences in these period are so hard to interpret. At no point in our paper do we use training data alone to define our effects. Our decision to show this data was based on our desire for full transparency and to provide proof that our DREADD manipulation was not drastically altering baseline locomotion or tone responsivity.

We maintain that without a significant difference in the recall tests, we cannot claim there was an effect of BA pyramidal neuron inhibition on learning, as defined through the traditional use of recall tests. However, we see how our previous wording of this section led to confusion regarding our claims. It is not our intention to obscure the presented data. We have re-worded this section based on the reviewer's suggestion: page 11, lines 60-64.

2. I appreciate the authors' inclusion of additional points in the Discussion, particularly regarding gating. However, the way authors use ‘gating’ and the way Luthi and others use the term are different. For Luthi (e.g., the Wolff 2014 paper the authors repeatedly cite), gating plays a permissive or enabling role for the synaptic plasticity underlying the memory formation. Thus, gating mechanisms can be manipulated to either enhance or impair the memory formation. Wolff et al. (2014) use CS-US pairings throughout the experiments (in contrast to the effects the authors of this manuscript find without a US) and demonstrate that PV neuron manipulations can enhance and impair the memory. Luthi's group argues that the PV neurons play a role in gating the CS-US associations.

In contrast, the authors of the present manuscript demonstrate that activation of BA neurons in the absence of a US can create a memory, yet such activation has no effect on a single CS-US pairing. At minimum, if this ‘gating’ were like Luthi's gating, such activation would enhance the memory formation of the single CS-US pairing. Considering the novelty of the authors' findings, the authors do themselves (and their findings) a disservice by suggesting that their findings are of the same kind as Wolff et al. The authors' findings are different and consequently rather interesting. I would suggest the authors refrain from referring to their findings as ‘gating’ or, at minimum, suggest that, if this is gating, it is different from what Wolff found in their work.

Please see full response below.

Reviewer 2:

This timely study with relevant findings extend an important literature on the study of associative learning mechanisms by highlighting a novel role for inhibitory signaling in the amygdala.

In this revised version, the authors addressed the concerns raised by making relevant changes to the text of the manuscript and figures. This changes included toning down the interpretations of their findings and thereby changing the title. Since there is no direct evidence for gating mechanism I suggest the authors mention this concept to a minimum.

As both reviewers have expressed justifiable concerns regarding the use of the term ‘gating’ to describe our effects, we have removed this section from the manuscript. We refer to our effects as ‘promoting’ fear memory formation/learning throughout: page 21, lines 87 and 95. Further, we have limited our discussion of the work by Luthi et al, which is now included only as further evidence supporting the role of pyramidal neuron excitation in the LA during fear memory formation: text removed from the Discussion section: page 21.